Fibrinogen is a soluble plasma glycoprotein which is synthesized in the human body primarily by liver parenchymal cells. It is a dimeric molecule, consisting of two pairs of three polypeptide chains designated A.alpha., B.beta. and .gamma., which are connected by disulfide bridges. The three polypeptide chains are encoded by three separate genes. The wild-type A.alpha. chain is synthesized as a 625 amino acid precursor and is present in plasma as a 610 amino acids protein, the B.beta. contains 461 and the .gamma. chain 411 amino acids. The three polypeptides are synthesized individually from 3 mRNAs. Assembly of the three component chains (A.alpha., B.beta., and .gamma.) into its final form as a six-chain dimer (A.alpha., B.beta., .gamma.)2 occurs in the lumen of the endoplasmic reticulum (ER).
Fibrinogen circulates in blood at high concentrations (1-2 g/L) and demonstrates a high degree of heterogeneity. Variations arise through genetic polymorphisms, differences in glycosylation and phosphorylations, (partial) proteolysis of the carboxy-terminal part of the A.alpha. chain and alternative splicing (for review see De Maat and Verschuur (2005) Curr. Opin. Hematol. 12, 377; Laurens et al. (2006) J. Thromb Haemost. 4, 932; Henschen-Edman (2001) Ann. N.Y. Acad. Sci. USA 936, 580). It is estimated that in each individual about one million different fibrinogen molecules circulate. Most of these variants, which account for just a small portion of the total fibrinogen (in most cases not more than a few percents), differ in function and structure. Proteolysis of the carboxy-terminal part of the A.alpha. chain results in three major circulating forms of fibrinogen having clearly different molecular weights. Fibrinogen is synthesized in the high-molecular weight form (HMW; molecular weight 340 kDa; the predominant form of A.alpha. chains in the circulation contains 610 amino acids). The degradation of one of the A.alpha. chains gives the low-molecular weight form (LMW; MW=305 kDa); the LMW′ form (270 kDa) is the variant where both A.alpha. chains are partially degraded at the carboxy-terminus. In normal blood, 50-70% of the fibrinogen is HMW, 20-50% is fibrinogen with one or two degraded A.alpha. chains (de Maat and Verschuur (2005) Curr. Opin. Hematol. 12, 377). The HMW and LMW′ variants show distinct differences in clotting time and fibrin polymer structure (Hasegawa N, Sasaki S. (1990) Thromb. Res. 57, 183).
Well-known variants which are the result of alternative splicing are the so-called .gamma.′ variant and the Fib420 variant.
The .gamma.′ variant represents about 8% of the total of .gamma.-chains. It consists of 427 amino acids rather than 411 for the most abundant .gamma.-chain; the four C-terminal amino acids (AGDV) are replaced by 20 amino acids that contain 2 sulphated tyrosines. The fibrinogen .gamma.′ chain is not able to bind to the platelet fibrinogen receptor IIb.beta.3, which is critical in regulating platelet aggregation.
The Fib420 variant, which has a molecular weight of 420 kDa, accounts for 1-3% of the total circulating fibrinogen (de Maat and Verschuur (2005) Curr. Opin. Hematol. 12, 377). Through alternative splicing, an extra open reading frame is included at the C-terminus of the A.alpha.-chain, thereby extending it with 237 amino acids. The additional amino acids form a nodular structure.
Plasma derived fibrinogen is an important component of marketed fibrin sealants which are clinically applied during surgical interventions to stop bleeding and to decrease blood and fluid loss. In addition it is used to facilitate tissue adherence by using the agglutination property of fibrin and to improve wound healing. Fibrinogen is also used clinically to supplement fibrinogen deficiency in hereditary fibrinogenemia patients and in patients with an acquired fibrinogen deficiency. Intravenous administration of high dosage of fibrinogen (3-10 gram) has demonstrated to normalize clotting of blood and arrest or prevent serious bleeding in various clinical situations.
Recombinant production of human fibrinogen, be it in wild-type (HMW) format or as a variant (e.g as Fib420), has many advantages over the use of plasma derived materials. These include its preferred safety profile, the possibility to make variants in a pure way and unlimited supply. However, in order to produce it in an economically feasible way, high expression levels of intact, functional fibrinogen are required. In addition, for specific applications (e.g. use of fibrinogen as an intravenous (IV) hemostat) proper post-translational modifications (e.g. glycosylation) are required.
Because of the post-translational modifications, expression in mammalian systems is preferred. Therefore, biologically active recombinant fibrinogen has been expressed in various cells, such as baby hamster kidney (BHK) (e.g. Farrell et al. (1991) Biochemistry 30, 9414), Chinese Hamster Ovary (CHO) cells (e.g. Lord, (U.S. Pat. No. 6,037,457), Binnie et al. (1993) Biochemistry 32, 107), or African Green Monkey derived COS cells (e.g. Roy et al. (1991) J. Biol. Chem. 266, 4758). However, the expression levels are only around 1-15 .mu.g/ml and considered inadequate to replace the large amounts of plasma fibrinogen needed in clinical practice. In addition, expression of human fibrinogen in yeast P. pastoris yielded 8 .mu.g/ml, which is also not adequate for commercial manufacturing (Tojo et al. (2008) Prot. Expr. and Purif. 59, 289).
In EP 1 661 989 it is reported that yields of at least 100 mg/L are needed for commercial viable production. In this application levels of up to 631.5 mg/L by CHO cells in a spinner flask are reported. However, in order to reach such levels, cells have to co-express the baculovirus P35 anti-apoptosis protein, and methotrexate, an anti-metabolite, has to be used for amplification of the vectors. Cell densities are relatively low (maximum in spinner flask 9.4.times.10.sup.5 cells/ml in 15 days) as compared to what is standard in the industry e.g. Wurm (Nature Biotechnol. (2004) 22, 1393) reports routine cell densities of 2.times.10.sup.6 cells/ml in 3-4 days of subcultivation).
The most important issue for the successful production of recombinant fibrinogen is how to make enough intact, properly assembled, biologically active product at high purity.